Fun 2: 11:00 - 12:00 Scribe: Kristina Hixson

Fun 2: 11:00 - 12:00 Scribe: Kristina Hixson

Monday, November 2, 2009 Proof: Matt Davis

Dr. Waites Mycoplasma & Fastidious Gram Negative Bacteria Page 1 of 8

I.  Introduction: Mycoplasma and Fastidious Gram Negative Bacteria [S1]:

a.  Going to talk about more bacteria, some of the ones we will talk about today we will see in lab tomorrow. Tomorrow’s lab is a 2 part lab. We will have the first part tomorrow and you are going to be setting up some things and looking at some things tomorrow and doing case studies. Then we will actually have you come back on Wednesday morning to look at the results of some of the things you are going to set up. There will be a lab report that you will turn in on Wednesday.

b.  This lecture is mycoplasma and fastidious Gram negative bacteria. We will talk about the Haemophilus, Bordetella, Legionella, Morexcella, Mycosplasma, Ureaplasma, Bruchella, and Francisella.

c.  Some of them that we are going to talk about, we will not spend a great deal of time individually on them, we will mainly talk about the most important things that you need to be familiar with for this misc. group of microorganisms. These are the bacteria that do not really fit into the other categories that we have talk about already, so we just put them all together as one group.

II.  Objectives [S2]

a.  I’m going to use the same format as I used last week when we talk about other groups of bacteria. We’ll talk about their characteristics, their epidemiology, how they produce disease, the diseases they produce, and how we would identify someone that had an infection with them.

III.  Haemophilus [S3]

a.  The 1st bacteria we will talk about and that you will meet tomorrow in lab is Haemophilus. If you look at the derivation of the name Haemophilus, heme meaning blood, philus meaning loving. So it is a blood loving organism. It likes blood because it has to have blood, or at least the components of blood cells, in order to live and proliferate.

b.  Haemophilus is a very small bacterium. It is a small coccobacillus, it is an intermediate. It is not round like a coccus and it is not long like a rod, it is intermediate and it is very small as you will be able to see on the Gram stain tomorrow. So here you see some in this smear here.

c.  Facultative anaerobe, non-hemolytic. So you won’t be able to see it at all on regular blood agar and it won’t even grow, so I refer to it as a fastidious organism, because it requires supplemented media such as chocolate agar, in which the blood has been heated so that it lyses the red blood cells. Many bacteria that produce the hemolysis can open red blood cells and get the things that they need outside of the cell, but Haemophilus is not able to do it. Haemophilus causes non-invasive disease as well as invasive diseases. One of its virulence factors is a capsule. There are several different capsule types. It is the encapsulated strains that tend to cause the most severe and invasive infections. The non-encapsulate Haemophilus influenzae and the other species of non-encapsulated Haemophilus may be found in the upper respiratory tract in healthy people.

IV.  Haemophilus Pathogenesis [S4]

a.  This is primary a respiratory pathogen and it disseminates by respiratory aerosols, like so many other bacterial and viral pathogens do.

b.  In some cases since it is prevalent in the upper respiratory tract for long periods of time you can get an endogenous infection where you get an activation or an invasive disease that occurs from bacteria that may be there already, but it is just that something has changed between the host and the microorganism’s relationship there.

c.  The capsule is the most important virulence factor; it serves the function of being antiphagocytic, like most other bacterial capsules do. There are 6 capsular types and they are named A-F. It is type B that is the one that is the most invasive and causes the most serious diseases there. Since it is a Gram negative bacterium, by definition it contains endotoxin in its cell wall. The endotoxin and the effects that this has locally is some of the things that actually helps damage the respiratory tract when this organism gets down into the lower respiratory tract and starts multiplying. This is what allows it to become bacteremic in some cases. It is local damage endotoxin that allows it to spread to the blood stream and cause more severe disease.

d.  This bacterium does not produce any notable exotoxin. It does produce an IgA protease to help protect it from secretory IgA. Another virulence factor that we see in about 30% of the strains and this has been holding true for the last several years, is that about a third of them will produce a beta lactamase enzyme. This is like the enzyme I told you about with the Staphylococcus. This is an enzyme that the bacteria produce that will actually destroy the beta-lactam antibiotics like the penicillin. So about a third of the time those types of antibiotics will not work on Haemophilus influenzae infections.

V.  Detection H. influenzae [S5]

a.  This is Haemophilus on a chocolate agar plate. So you see chocolate agar, but no growth on blood agar or MacConkey agar and you will see these things in lab tomorrow.

b.  It is a cathophilic organism, it requires CO2.

c.  One of the other things that we can use to actually distinguish Haemophilus influenzae from some of the other Haemophilus species, of which there are several, is the differential requirements of the X and the V factors. We have a demonstration of this in the lab tomorrow for you to appreciate what I am talking about.

d.  The X and the V, don’t get them confused with the 5 and the 10, which is the coagulation factors, we talk about those. The X is heme and V is NAD. These are cofactors. Heme is from hemoglobin iron pigment. The NAD is the enzyme cofactor and these are things that are found inside red blood cells. So hemolytic bacteria that need these nutrients can get them when they hemolosize the red cells. Since the Haemophilus influenzae can’t hemolyze red blood cells, unless you put it on the chocolate agar where the cells have been lysed. You have to supply it. So you take a non-supplemented media such as Mueller-Hinton agar that doesn’t have these nutrients and you can put them on a filter paper disc and you can steak the plate out with Haemophilus and you can see where the organisms will grow. In this particular case, you see growth around the X and the V together. You can see the haze here, the cloudiness here is growth of the bacteria, where you don’t see any growth around each of these by themselves.

e.  Some of the other species of Haemophilus, which are less pathogenic or normal flora organisms that you are wanting to differentiate from Haemophilus influenzae, is that they may require one or the other, but not necessarily both.

f.  One of the other things you see with H. influenzae that we will have set up in the lab tomorrow is whether or not it will produce satellitism. Staphylococcus aureus is a strongly beta hemolytic organism, remember if you can lyse the red cells you can get the X from the V, so the way you can see if you’ve got a Haemophilus you can see is that you have satelliting. You can take a regular blood agar plate that Haemophilus will not normally grow on and streak the Haemophilus across the blood agar plate and then take a Staphylococcus aureus and streak it down the middle of the plate. The area where the Staph produces the hemolysis you may see some of the little small colonies of Haemophilus going around the Staph because it can only grow on that part of the blood agar plate where the Staph has lysed the red blood cells. So it will satellite around that Staph strain there and that is what we see satellitism. We use the in the clinical laboratory sometimes to identify this organism. This is the classic way you describe Haemophilus influenzae. We actually use a variety of different biochemical systems to identify Haemophilus now. We don’t use this is in the lab because this takes an over night test and we have a quick biochemical test that we can do in 4 hours but this is the test that you use to identify the organism and distinguish it from some of the others.

VI.  Diseases H. influenzae [S6]

a.  Some of the diseases H. influenzae has been associated with are mainly respiratory tract mediated, but of course they can become systemic. Usually it starts in the respiratory tract.

b.  Middle Ear infections: otitis media is important. H. influenzae is an important cause of this especially in children. It can cause sinusitis.

c.  H. influenzae historically has been the most important cause of epiglottitis. It is almost always a type B H. influenzae. Epiglottitis, as the name, would imply is an inflammation of the epiglottitis, which is the little flap that goes over the trachea to keep you from aspirating food as you inhale there. If the organism that’s present in the respiratory tract grows and causes an infection of the epiglottitis it can be a life threatening emergency especially in young children because edema of the epiglottitis, can close and obstruct the trachea and cause affixation. Laryngotracheal bronchitis as the name implies is an inflammation of the upper and lower airways in severe cases historically. I will talk about the vaccine a little bit more. We’ve had a vaccine for Haemophilus type B influenza since the 1980’s. Since the Haemophilus vaccine has been in place as part of the routine childhood immunization protocol, epiglottitis is almost never seen anymore. Meningitis is decreased by about 95%, so you hardly ever see meningitis due to Haemophilus anymore. In the past meningitis with Haemophilus was very important in older infants and young children. There is protective maternal antibody that is passed through the placenta that protects the very young infants from diseases like this, but remember that maternal antibody begins to give way after a few of months because they’ve already made more to take its place. But then young children will be at risk for Haemophilus meningitis. So in children, a year and a half to two it is very important cause of meningitis, which we don’t see anymore.

d.  We do see it as the cause of community acquired pneumonia. One of the most common things that we see as Haemophilus influenzae disease now is in middle age to older people with chronic obstructive pulmonary disease which emphysema or chronic bronchitis, usually related to smoking. They start producing a lot of secretions and have fever and then you’ll grow Haemophilus in large amounts from their sputum. Because it is often present in the respiratory tract normally, something goes wrong to upset the balance between the organisms and the host that starts multiplying in the setting of an underlying lung disease and you get an exacerbation of it.

e.  Pneumonia I’ve already mentioned that

f.  This little girl has cellulitis of the face due to Haemophilus influenzae. We’ve already mention otitis media

g.  Conjunctivitis is very important for the optometrists, when people come in with a red eye. Haemophilus influenzae and Haemophilus aegyptius which is a very similar organism, these are fairly common causes of bacterial conjunctivitis. So anything around the face and the upper respiratory tract can certainly be a problem with this.

VII.  Prevention H. influenzae [S7]

a.  The vaccine is important. Invasive disease is rare in people over age 5 even without the vaccine because you start producing antibodies because you encounter the organism frequently. The problem with children after the time when maternal antibody would depart before they developed their own antibody repertoire, the toddlers and the very young preschool children were the ones who got the epiglottitis, bacteremia, and meningitis. So in the mid 1980’s a vaccine was developed against type B which is the invasive type. The non-encapsulated types don’t usually cause the severe disease, we still see them as cause of infections because they are not covered in the vaccine, but this was a polysaccharide capsule vaccine that was coupled to a protein carrier to help to stimulate/boost its immunogenic effect in varying children. 3-4 does is now given in infancy or early childhood and we have a significant reduction in the disease due to type B, which is the invasive type. So this is a real success in terms of vaccines and public heath in the prevention of Haemophilus infections.

VIII. Other Haemophilus species [S8]

a.  Here you see a Haemophilus conjunctivitis.

b.  Here you see (this is the tip of the urethra) and you see a genital ulcer. A chancroid is a disease caused by an organism that we formally know as Haemophilus ducreyi. It is a painful genital ulcer that could be theoretically confused with the chancre that you get from syphilis. The difference between this chancre and the syphilitic chancre is that the syphilitic chancre is painless and this one is quite painful. So chancroid is a disease caused by Haemophilus ducreyi

IX.  Bordetella pertussis [S9]

a.  The next organism we will mention is Bordetella pertussis.

b.  It is an encapsulated, gram negative coccobacillus that looks like this. It is a slow growing fastidious, strict aerobe. It is fairly metabolically inert. It doesn’t ferment carbohydrates, but it does oxidize amino acids.

X.  B. pertussis: Whooping Cough [S10]

a.  It produces whopping cough.

b.  If we go to this website then we can actually hear the cough. This is a website that has the audio with a child with whopping cough. See they cough several times and then they can’t breathe while they are coughing so when they stop they have to take a big inspiration and that is the whoop.